Fig. 5. YtfK can interact with SpoT and trigger (p)ppGpp synthesis.

a YtfK interacts with SpoT in BTH assays. Briefly, overnight culture of BTH101 strain harboring pUT18c ytfK (or pUT18c ytfK^P42L) and pKT25-spoT (or pKT25 relA) were spotted on X-Gal agar base plates (see the Methods section). The blue color indicates a positive interaction. The results of the β-galactosidase assays using the same strains are shown on the horizontal graphs on the right. Error bars indicate the SDs of averages of three independent experiments. b In vivo (p)ppGpp accumulation following ectopic expression of both ytfK and ytfK^P42L. WT strain or ΔrelA mutant carrying ytfK or ytfK^P42L on pEG25 were grown exponentially in phosphate MOPS minimal medium (see the Methods section). Samples were collected before and after ytfK induction (1 mM IPTG) prior to nucleotides extraction and were then separated by TLC. Representative autoradiograph of the TLC plates is shown and quantification is provided in (c). Error bars indicate the standard deviations of averages of three independent experiments. d The intracellular level of (p)ppGpp induced by ytfK overexpression controls the growth rate. Growth curve of WT (red) strain or ΔrelA mutant (blue) carrying ytfK (solid lane) or ytfK^P42L (dashed lane) on pEG25 in the LB medium. Overnight culture was diluted 100 times, and growth was monitored at 600 nm. At the indicated time, 1 mM of IPTG was added to induce ytfK expression. Error bars indicate the standard deviations of averages of three independent experiments. Source data are provided as a Source Data file.